Throughout this application, various publications are referred to within brackets. The disclosures for these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The discovery of catalytic RNA molecules that possess enzymatic, self-cleaving activity (ribozymes) has provided a new way to artificially control gene expression (Foster & Symons, (1987) Cell, 49:585-591). Ribozymes have been designed that contain sequences required for the cleavage of particular RNA sequences. For the hammerhead type of ribozyme, the target RNA needs to contain only the sequence XUX with cleavage occurring 3' from XUX (Haseloff & Gerlach, (1988) Nature, (London) 334:585-591; Perriman et al., Gene (1992) 113:157-163). High specificity and limited target requirement give these catalytic RNA molecules the potential to inhibit viral pathogens and to regulate specific gene expression by interfering with transcription in a highly specific manner (Uhlenbeck, (1987) Nature (London) 328:596-600; Haseloff & Gerlach, (1988) Nature, (London) 334:585-591).
Several reports indicate that a hammerhead type of ribozyme functions in living cells. Cotten & Birnstiel (1989, EMBO J., 8:3861-3866) and Cameron & Jennings (1989, Proc. Natl. Acad. Sci., USA 86:9139-9143) have reported ribozy-memediated destruction and an inhibition of specific gene expression in Xenopus laevis oocytes and monkey (COS1) cells. Sarver et al. (1990, Science, 247:1222-1225) showed that a ribozyme directed against HIV-1 gag RNA reduced p24 antigen expression in CD4.sup.+ HeLa cells. Recently, this line of study was extended to bacterial cells by showing that a ribozyme designed to cleave the integrase gene of HIV-1 is effective when transcribed from a plasmid in Escherichia coli. Integrase RNA was eliminated and integrase protein synthesis was blocked (Sioud & Drlica, (1991) Proc. Natl. Acad. Sci., USA 88:7303-7307). Since ribozymes are effective in vivo, problems of ribozyme stability and delivery may now be addressed.
To interfere with tumor necrosis factor-.alpha. (TNF-.alpha.) gene expression we have used cationic liposome-mediated transfection (Malone et al., (1989) Proc. Natl. Acad. Sci., USA 86:6077-6081) to deliver a ribozyme directed against TNF-.alpha. into human promyelocytic leukaemia cells (HL60) and peripheral blood mononuclear cells (PBMNC). TNF-.alpha. plays an important role in many inflammatory rheumatic diseases (Shinmei et al., (1989) Sem. Arth. Rheum. 18 (suppl. 1) 27-32), and it modulates the expression of several proteins, including the Class I antigens of the major histocompatibility complex (MHC) and cytokines such as interleukin-1 and interleukin-6 (Beutler & Cerami, (1988) Annu. Rev. Biochem. 57:505-518 and (1989) Annu. Rev. Immunol. 7:625-655). TNF-.alpha. also appears to be necessary for normal immune responses, however large quantities of it can produce destructive effects such as rheumatoid arthritis (Brennan et al., (1989) Lancet ii 244-247). TNF-.alpha. is the cytokine responsible for the induction of HIV-1 expression in ACH-2 cells (Rosenberg & Fauci, (1990) Immunol. Today 11:176-180). TNF-.alpha. induces the production of cellular factors that bind to the NF-KB enhancer elements within the viral long terminal repeat sequences and thereby activates HIV-1 expression.
The effectiveness of catalytic RNA molecules is dependent on the stability of the mRNA in vivo. Compared to the current knowledge of DNA structural elements, relatively little is known about mRNA stability elements. mRNA half-lives range from less than 30 minutes for fibroblast interferon and c-fos to greater than 17 hours for .beta.-globin MRNA. Most eukaryotic mRNAs are protected from exonuclease attack by the 5' cap structure and the 3'poly(A) tail and poly(A) binding proteins. Eukaryotic mRNAs have both 5' and 3' non-coding regions on either side of the coding region. The 5' non-coding region is involved in the rate of initiation of translation of the mRNA to protein. The 3' non-coding region serves to initiate the formation of the poly(A) and can act to stabilize mRNA (Baralle, F. E., Int. Rev. of Cytology (1983) 81:71-106). In particular, 3' non-coding iron-responsive elements have been identified that can modulate mRNA stability in the presence of iron. Another characterized motif is the AUUUA element responsible for the rapid degradation of some cellular mRNAs, particularly cytokine mRNAs. (Saini, K. S. et al., Mol. Cel. Biochem. (1990) 96:15-23; Ross, H. J. et al., Blood (1991) 77:1787-1795). Some have postulated that an initial endonuclease attack is required before rapid degradation can take place (Nielson, D. A. and Shapiro, D. J., Mol. Endocrinology (1990) 4:953-957).
There is a need for a method to extend the half-life of particular mRNAs in vivo and increase protein levels as well as to control and reduce gene expression with oligonucleotides (e.g., antisense and triple helix) in plants and animals. The stabilization of mRNA elements can be effected through the use of ribozymes as well as antisense oligonucleotides.